Normal peripheral blood contains mature red blood cells which are free of nucleus and reticulum. Nucleated red blood cells (NRBCs), or erythroblasts, are immature red blood cells. They normally occur in the bone marrow but not in peripheral blood. However, in certain diseases such as anemia and leukemia, NRBCs also occur in peripheral blood. Therefore, it is of clinical importance to measure NRBC. Traditionally, differentiation and enumeration of NRBC are performed manually. The process involves the smearing of a blood sample on a microscope slide and staining the slide, followed by manual visual analysis of the individual slide. The NRBC concentration is reported as number of NRBCs per 100 white blood cells. Usually, 200 white blood cells and the number of NRBCs present in the same region on a blood smear are counted and the numbers are divided by two to express the NRBC concentration as the number of NRBCs/100 white blood cells. This approach is extremely time-consuming as well as being subjective to the interpretation of the individual analyzing the slide.
In recent years, several fluorescence flow cytometry methods have been developed for differentiating NRBCs. These methods utilize specific nuclear staining technique to distinguish NRBCs from other cell populations because it is not easy to differentiate NRBCs based on their electronic or optical properties.
U.S. Pat. No. 5,298,426 (to Inami et al.) discloses a fluorescence method for differentiating NRBCs. The method utilizes a two-step staining using a first fluid which is an acidic hypotensive fluorescent dye solution, and a second fluid which changes the osmolality and pH of the first fluid. Inami et al. teaches that the first fluid contains an erythroblast-staining dye that diffuses into nucleated red blood cells to specifically stain their nuclei, and then separating a group of NRBCs from other cell groups on a two-dimensional plot whereby the results of NRBC differentiation are computed. In order to differentiate leukocyte subpopulation concurrently, the first fluid also contains two additional fluorescence dyes, i.e., an eosinophil/basophil-staining dye and a leukocyte-staining dye for specific staining of these cell types.
U.S. Pat. No. 5,559,037 (to Kim et al.) discloses a method for flow cytometric analysis of NRBCs and leukocytes. The method comprises lysis of red blood cells and NRBC cytoplasm from a whole blood sample to expose the NRBC nuclei to a vital nuclear stain and minimizing the permeation of the vital nuclear stain into the leukocytes and analyzing the sample by measuring fluorescence and two angles of light scatter. This method features a triple triggering method which blocks the signals from debris (fluorescent and non-fluorescent) and identifies the signals which fall below the ALL trigger but above the fluorescence trigger (FL3) as NRBCs. ALL is the axial loss of light or the light scatter signals detected at 0.degree. from the incident light. Therefore, pre-gating of signals in more than one dimension is required in this method for identification of NRBC population. Since leukocytes are also nucleated cells, staining of these cells needs to be prevented to avoid interference to the fluorescence measurement. The preservation of leukocyte membrane and minimizing the permeation of the nuclear stain into the leukocytes are achieved by concurrently fixing the leukocytes with an aliphatic aldehyde during lysis of red blood cells. The aldehyde fixatives are known as hazardous chemicals. In addition, the method requires heating of the reagent to 42.degree. C. in order to obtain the NRBC and leukocyte differentiations.
U.S. Pat. No. 5,631,165 (to Chupp et al.) discloses an automated method for counting and differentiating nucleated red blood cells or reticulocytes and cell surface antigens in a lysed blood sample. The method detects multi-angle light scatter and fluorescence signals from the lysed blood sample in a flow cell. Similar to U.S. Pat. No. 5,559,037 (to Kim et al.), this method requires detection of fluorescence signals from nucleated red blood cells or reticulocytes produced by nuclear staining with a fluorescent dye.
The above-described methods are able to differentiate and enumerate NRBCs and leukocytes by fluorescence flow cytometry. However, fluorescence measurement is a complex and expensive detection method.
Current automated hematology analyzers, such as Abbott Cell-Dyn.RTM. 3500, COULTER.RTM. STKS.RTM., Technicon H*1.RTM. and TOA Sysmex.TM. NE-8000 are only able to provide NRBC flagging for the possible presence of NRBCs in an analyzed blood sample when the instruments sense an increased amount of signals near red blood cell debris area of a histogram. However, such techniques frequently generate false positive flagging because many other blood abnormalities can cause increased signals at the same area, such as platelet clumps and sickle cells, as well as red cell debris from insufficiently lysed blood samples. In these methods NRBCs are not identified. Instead, only a common NRBC sample distribution pattern in a histogram or a dotplot is recognized by the instrument which can be easily confused with a similar pattern generated by above-mentioned other causes. For the flagged samples, including false positive flags, re-examination of the sample with manual method is required in clinical laboratories. Another problem with the NRBC containing samples is that the white blood cell count (WBC) reported by hematology analyzers is not accurate for these samples since NRBCs could elevate the WBC by being misidentified as white cells. On the other hand, analysis of leukocyte populations from whole blood samples is an integral part of diagnostic procedures regarding a multiplicity of pathologies. The ability to analyze the major subpopulations of leukocytes in an automated manner is essential for a rapid diagnosis of a single blood sample and for the rapid processing of many samples at once.
U.S. Pat. No. 5,155,044 (to Ledis et al.) discloses a method for isolation and analysis of leukocytes from a whole blood sample, which enables differentiation of leukocytes into five subpopulations in a one-step measurement on an automated hematology analyzer. The detection technique involves a concurrent light scatter measurement and impedance measurements in both DC (direct current) and RF (radio frequency). This method is simple and fast, but it does not provide differentiation of NRBCs.
U.S. Pat. No. 5,384,549 (to Hamaguchi et al.) describes a lytic reagent system and a method for differentiation of leukocytes into five subpopulations by a complex procedure. The method requires three lytic reagents, three separate sample preparations and measurements for the identity of eosinophil, neutrophil and basophil populations in addition to the lymphocyte and monocyte populations. Hamaguchi et al. describe merely the observation of abnormal leukocyte populations and nucleated red blood cells using the lysis reagent system and DC vs. RF detection method. However, this method is limited to only observing the presence of some abnormal cell types, but is not able to differentiate or enumerate the NRBCs.
U.S. Pat. No. 5,686,308 (to Li et al.) describes a lysing reagent system and a method for differentiation of leukocytes into five subpopulations in a one-step measurement on an automated hematology analyzer. The lytic reagent system comprises a lytic reagent comprising an ethoxylated long chain amine compound and acid to adjust the pH of the lytic reagent to be within the range of 2.0 to 3.6; and a hypertonic, alkaline stabilizing reagent. This patent teaches a reagent and method for differentiation of leukocyte subpopulations, but does not teach differentiation of nucleated red blood cells.
Based on foregoing, there exist a need for a simple and less costly analysis method for differentiating and enumerating NRBCs.